VOL. 52, 1964 GENETICS: FREESE AND FREESE 1289 8 Matthaei, J. H., and M. W. Nirenberg, these PROCEEDINGS, 47, 1580 (1961). 9 Wood, W. B., and P. Berg, these PROCEEDINGS, 48, 94 (1962). 10 De Moss, J. A., and G. D. Novelli, Biochim. Biophys. Acta, 18, 592 (1955). 11 Nathans, D., and F. Lipmann, these PROCEEDINGS, 47, 497 (1961). 12Raacke, I. D., S. Matsushita, and J. Fiala, unpublished. 13 Webster, G., and S. L. Whitman, Biochim. Biophys. Acta, 68, 653 (1963). 14 Levinthal, C., A. Keynon, and A. Higa, these PROCEEDINGS, 48, 1631 (1962). 15 Revel, M., and H. H. Hiatt, these PROCEEDINGS, 51, 810 (1964). 16 Weisberger, A. S., S. Armentrout, and S. Wolfe, these PROCEEDINGS, 50, 86 (1963). 17 Hardesty, B., J. J. Hutton, R. Arlinghaus, and R. Schweet, these PROCEEDINGS, 50, 1078 (1963). 18 Watson, J. D., Science, 140, 17 (1963). 1" Allen, D. W., and P. C. Zamecnik, Biochim. Biophys. Acta, 55, 865 (1962). 20 Cammarano, P., G. Guidice, and G. D. Novelli, Biochem. Biophys. Res. Commun., 12, 498 (1963). 2"Lederberg, S., B. Rotman, and V. Lederberg, Biochem. Biophys. Res. Commun., 12, 324 (1963). TWO SEPARABLE EFFECTS OF HYDROXYLAMINE ON TRANSFORMING DNA ]3 ELISABETH BAUTZ FREESE AND ERNST FREESE LABORATORY OF MOLECULAR BIOLOGY, NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS, NATIONAL INSTITUTES OF HEALTH Communicated by M. R. Irwin, October 1, 1964 In earlier experiments we observed a dual effect of hydroxylamine (HA) on phage T4. At high concentrations of HA (about 1 M) phages were readily mutated under conditions of little inactivation, whereas at lower concentrations (10-2 M) phages were rapidly inactivated without any induction of mutations.1 The muta- genic effect was shown to result from the specific reaction of HA with cytosine.' The highly inactivating effect, however, remained partially unexplained. It could be exclusively caused by the inactivation of phage tail protein, which has been shown to occur,3 or it could be due to a combination of two reactions, one on the phage tail and the other on phage DNA. In order to find out whether DNA was actually subject to a strong inactivation by low HA concentrations, we used the transformation system of B. subtilis, which allows one to measure both inactivation and mutation directly on the same DNA. The present paper will show that HA indeed exerts two different effects on DNA, a predominantly mutagenic effect at high HA concentrations (about 1 M) and a predominantly inactivating effect at low HA concentrations (102 to 104 M). From experiments with HA analogues it was furthermore possible to decide which group of HA is responsible for the "mutagenic effect" and which one is required for the "inactivating effect." Materials and Methods.-Transformation: DNA donor strain: 60 009 (=SB 19 of Romig) = prototroph. Recipient strain: 60 087 (= T, of Anagnostopoulos) = tryptophan . Transforma- tion to tryptophan independence and production of fluorescent mutants were assayed as described previously.4,' Each time-dependent curve was determined with the same batch of transformable bacteria. Downloaded by guest on October 1, 2021 1290 GENETICS: FREESE AND FREESE PROC. N. A. S. Chemicals: HA X HCl was bought from Eastman; 0-methyl-HA X HCl (= methoxyamine) and N-methyl-HA X HCl were obtained from the Aldrich Chemical Co., Milwaukee, Wis. The purity of the latter was checked by paper chromatography in 1:1 n-butanol: 6 M HCl on Whatman #1 paper. The paper was sprayed by a 0.5% solution of picryl chloride in ethanol and then ex- posed to ammonia vapor. The Rf values are 0.50 for HA and 0.69 for N-methyl-HA. By over- loading the paper it could be established that the impurity of HA in N-methyl-HA was less than 1%. pK values of HA derivatives were measured at a concentration of 10 mg/ml in a radiometer recording pH meter. Sodium hyponitrite was prepared by Alfa Inorganics, Inc., Beverly, Mass., and showed a pre- cise three-electron reduction with potassium permanganate. The UV spectra of dilute solutions agreed with results described in the literature.6 The absorption maxima were observed for 2450 A at pH 12, 2280 A at pH 9.9, and 2080 A at pH 3.5. The solution was stable at 250C (pH 3.5) but not after prolonged treatment at 750C. Treatment of DNA: DNA (130 ,jg/ml) was diluted tenfold in the ice-cold reaction mixture, mixed, and a control sample diluted 50-fold into ice-cold stopping mixture (0.05 M Tris pH 7.5 + 1 M NaCl + 10% acetone). The reaction mixture contained, in addition to the HA com- pound, 0.02 M Na-phosphate (at pH > 6) or Na-phosphate + succinate (at pH < 6), and NaCI to give the desired Na+ concentration wherever that has been especially noted. The reaction tube was then placed (about 30 sec after mixing) in a water bath at the desired temperature, usually 750C. At different times samples were diluted 50-fold into the stopping mixture. The DNA was then further diluted (1:10) in the transformation experiment. Results.-Induction of mutations: We discuss the mutagenic effect of HA before tackling inactivation, because the mutation results can be simply explained in terms of a single reaction mechanism. The induction of mutations was measured by the method of linked mutations described earlier.4 The recipient strain of B. subtilis was a mutant lacking the tryptophan synthetase activity; it could grow on tryptophan but not on indole. The prototroph donor DNA was treated by HA under various conditions and added at suboptimal (but constant) concentration to bacteria in the transformable t06 o 2M) 424H 4OUS m ota O 2 3 5 6 7 o os S FIG. 1.-Induction of fluores- FIG. 2.-Rate of mutation induction at cent mutants at different HA different concentrations of HA and its concentrations. pH 6.2, 750C. derivatives. Temperature 75°. n = HA, * = 2 MHA; as = 1 MHA- pH4.2- = HA, 3M Na, pH6.2- A- o = 1 M HA + 3 M Na+; HA, pA 6.2; *-N-methyl-HA, pH 6.2; * = 0.5 M HA; o = 0.5 M * = carboxymethoxyamine, pH 6.2; 0 = HA + 3 M Na+; A 0.1 M HA, 0-methyl-HA, pH 6.2, Downloaded by guest on October 1, 2021 VOL. 52, 1964 GENETICS: FREESE AND FREESE 1291 state. When these bacteria were plated on H suboptimal concentrations of indole, the fre- N-OH hydroxylanine quency of fluorescent mutants among trypto- H phan-independent transformants could be di- H rectly counted on the plates. \-OH N-thyl-hydroxylanino During the treatment of DNA with HA, the / frequency of fluorescent mutants increased 3 linearly with time, as shown in Figure 1. H When the rate of mutation induction (% mu- N-O-CH3 0-methyl-hydroxylmine= tants induced/hr) was plotted against the con- / mothoxyamine centration of HA, a linear curve was obtained FIG. 3.-Structure of HA and some (Fig. 2). These two results indicate that each derivatives. mutation was established by a single chemical event. Figure 2 also shows that the rate of mutation induction increased with decreasing pH,4 but did not depend on the concentration of sodium ions. At HA concentra- tions lower than 0.1 M no induction of mutations could be detected. For example, the mutation rate was less than 0.1 per cent per hour at 10-5 M HA and pH 6.2. Both N-methyl- and O-methyl-HA also induced mutations whose frequency in- creased linearly with time and analogue concentration (Fig. 2). The structures of these HA derivatives are shown in Figure 3. Inactivation: The inactivation of transforming DNA was measured by the de- crease in the number of tryptophan-independent transformants with the time of DNA treatment by HA or its analogues. The logarithm of the surviving fraction (B/Bo, with B = titer of transformants at time t and Bo = titer at time 0) de- creased linearly with the time of treatment, for HA concentrations up to 1 M, and for different temperatures, pH, or ionic strength. An example is shown in Figure 4. Only at 2 M HA did the rate of inactivation increase slightly with the time of treat- ment. We define here the inactivation rate k by k = d In Bo (t = time of treatment in hours). At 1 M HA the inactivation rates rapidly increased with the temperature, according to the Arrhenius equation, as can be seen from Figure 5. In order to obtain suffi- cient inactivation within a few hours of treatment, all of the following experiments were performed at 750C. Although the time dependence of inactivation seemed to indicate that a single chemical reaction caused the inactivation at each HA concentration (except at 2 M HA), the concentration dependence of k revealed the existence of two inactivating effects (see Fig. 6). Instead of a linear decrease of the inactivation rate with de- creasing HA concentration (which would give a 450 slope in Fig. 6), the value of k actually increased at pH 6.2, when the HA concentration decreased from 1 M. This was especially pronounced when the reaction mixture contained 3 M Na+ ions (see Fig. 6). At very high HA concentrations the curve bent upward again, obviously because the mutagenic effect, which increased with the HA concentration, exhibited a significant lethal effect. (It should be obvious that mutagenic reactions can be lethal if they occur in genes vital for the growth of the organism; the transforming pieces carrying the tryptophan markers apparently contain such vital genes.) At Downloaded by guest on October 1, 2021 1292 GENETICS: FREESE AND FREESE PROC.